WO2008098569A2 - Biologically active molecules, particularly based on pna and sirna, method for the cell-specific activation thereof, and application kit to be administered - Google Patents

Abstract

The aim of the invention is to produce biological active substances which can be introduced into cells both in vitro andin vivo and controllably inhibit gene expression exclusively in the desired target cells without influencing protein formation in other cells of the organism. Said aim is achieved by active substances (1) comprising at least one covalent bond to one or more peptides (2, 3), each of which has one or more specific amino acid sequences that are selected in respect of enzymes typical of the target cells. Said bond, which is broken exclusively by said enzymes typical of the target cells in order to biologically activate the active substance, allows the molecules to remain biologically inactive in cells other than the target cells. Said molecules are used for influencing gene expression of preferably sick and infected organs or cells, for example.

Description

 I P2376 Patent and Law Firm Bock- Bieber- Donath, Hans-Knöll-Str. 1, 07745 Jena |

Biologically active molecules, in particular based on PNA and siRNA, methods for their cell-specific activation and application kit for administration

The invention relates to specific biologically active molecules, in particular based on "peptide nucleic acids" (PNA) and "short interfering RNA" (siRNA), a method for transfecting them into a target cell and in this target cell, or immediately before transfection cell-specific, and an application kit for administration in conjunction with a transfection system. The said biologically active molecules interact after activation with the mRNA of the target gene and in the case of siRNA together with specific endoribonucleases form an RNA-protein complex called "RISC" (RNA induced silencing complex) .The RISC complex binds to the target gene. In this way, gene expression is inhibited and thus the formation of target proteins is inhibited In the case of the use of activated PNA molecules, binding to the target mRNA prevents translation. biologically active molecules can be used, for example, for controlling and inhibiting the growth of abnormal cells, in particular in the treatment of tumors, in the treatment of viral infections, in age-specific treatments, etc. In general, the cell-specifically activatable biologically active molecules can be used to modulate the gene expression of the target cells be used. Not only can the expression of genes be reduced, but they can also be increased by achieving a reduction in the expression of the negative regulator of the target gene by the biologically active molecules.

The inhibition of gene expression by introducing short (19-23 bp), double-stranded RNA molecules (siRNA) or PNA molecules into eukaryotic cells which is specific for a sequence segment of the mRNA of a target gene has already been described (Elbashir SM et al .: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411 (6836), 494-8; Liu Y et al .: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb 24, 43 (7), 1921-7; US 5,898,031; US 7,056,704).

Such molecules do not prevent the reading of a gene and the production of an mRNA, but in the case of siRNA, a cell-specific mechanism is initiated which degrades the target mRNA. Finally, as described above, the formation of a specific protein is suppressed without affecting the expression of other genes (post-transcriptional gene silencing).

In order to suppress the expression of a gene, the siRNA and PNA molecules can be introduced directly into the cell, in particular via transfection reagents and electroporation (Zhang M et al .: Downregulation-enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1 (1), 74-7; Gilmore IR et al .: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22, Curr Drug Deliv. 2006 Apr, 3 (2), 147-5; US 6,506,559).

The disadvantage here is that the siRNA is relatively unstable, which can be improved by chemical modifications (US 6,107,094).

A special problem of the therapeutic application of biologically active molecules is an application in vivo. For such

Application possibilities have been developed to stabilize the siRNA to reduce degradation (Morrissey et al .: "Chemical

Modifications of Synthetic siRNA ", Pharmaceutical Discovery, May 1,

2005), and transfection reagents, such as nanoparticles, have been developed in vivo jetPEI ™ (Polyplus), which also introduce the siRNA into cells in vivo (Vernejoul et al .: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer modeis, Cancer Research 62, 2002, 6124-

31; Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of polyethyleneimine (PEI) -complexed siRNA in vivo, Gene Ther 12 (5),

2005, 461-6.). Also, methods have been developed to transfect cells of a target tissue with siRNA in vivo (Ikeda et al .: "Ligand-Targeted Delivery of Therapeutic siRNA", Pharmaceutical Research, Vol 23, No. 8, August 2006).

The administration of biologically active substances in vivo, however, is often problematic due to the systemic effect. The introduction of these substances selectively in target cells is not sufficiently specific. This is particularly disadvantageous for siRNA and PNA molecules which are intended to be selective and exclusive in target cells. By tissue- or cell-specific-labeled transfection reagents (eg Antiköφer / antigen-labeled

Nanoparticles, TAT protein flanking, u. a.), no sufficiently large cell specificity is achieved. Miscar transfections are the result.

Furthermore, it is known to inactivate siRNA molecules by binding fluorochromes in their biological activity and to convert them back into their active form by irradiation with light of a specific wavelength (QN Nguyen et al .: Light controllable siRNAs regulate gene suppression and phenotypes in cells, Biochim Biophys Acta, 2006). This activation is initiated from the outside and is in no way cell-specific. As a result, the said siRNA molecules after their activation in turn also act unintentionally in all other transfected cells and not only in the intended target cells.

In addition, this mechanism is also difficult to apply in vivo.

The invention is based on the object to provide biological active substances which are transfectable both in vitro and in vivo in a target cell and inhibit there exclusively the expression of genes, without in other cells of the organism, the drug-specific expression of the target gene and thus the formation of proteins. For the exemplified treatment of tumors, this means the expression of the target gene and thus the protein formation in To selectively inhibit tumor cells, without affecting healthy cells, which can also be achieved by the active ingredients, as well as their survival.

According to the invention, the biologically active molecules, in particular PNA and siRNA, are covalently bonded to one or more peptides, each of which has at least one amino acid sequence selected in each case for target cell-typical enzymes and respectively for the covalent bond and its break-up. This covalent bond inactivates the biologically active molecules. There is thus no inhibition of specific gene expression after transfection into cells, as long as only one of the bound peptide chains remains on the PNA or siRNA molecules by the absence of the corresponding target cell-typical enzyme.

By means of a suitable transfection system, for example nanoparticles or sheath molecules such as liposomes, the inactivated active substance molecules can be transfected into the target cells. There, said inactivating covalent bonds are cell-typically disrupted by the cell-specific enzyme (s) relevant to the amino acid sequences of the coupling peptide or peptides, thereby activating the biological activity of the molecule now in the target cell and dissolved by peptides. This then binds to the specific mRNA of the target cell and thereby inhibits gene expression in a known manner in this particular cell.

In all other cells of the organism than the predetermined target cells into which the said inactive molecular constructs can also enter, the active agent molecules remain inactive, since the covalent bonds between the biologically active molecule, in particular PNA and siRNA, and the peptide (s) are due to the absence of the or the target cell-specific enzymes completely (no peptide bond was broken) or partially (not all peptide bonds were broken) remain. The biologically active molecule is due to the still covalent Peptide binding no binding with the mRNA of this cell, or it is in the case of using siRNA no RISC initiated.

Although, for example, in the treatment of tumors, the molecule constructs according to the invention to be transfected in their inactive (bound) form are not only in or on tumors

Target cells reach, but (which can hardly be avoided in practice) can also reach healthy cells are selectively exclusively in or on the tumor-targeted cells with the local cell-specific enzymes that said molecule in its biological

Activated and inhibited the drug-related expression of the target gene. This gene expression and thus the protein formation for the

The survival of the healthy cells remains despite the non-diseased (or intact for the intended biological effect) located cells, but permanently inactive

Molecular constructs unaffected by this drug.

It is also possible to activate one or more of these covalent bonds already partially before the transfection into the target cell by one or more cell-specific enzymes present on their surface or in their environment (based on the total number of covalent peptide bonds). For example, multiple and each directed to different enzymes of the target cell peptide chains could be present with amino acid sequences of which one or more bonds are already broken directly on the surface of the target cell or in their environment.

It would also be conceivable that the biologically active molecules in the state inactive by said peptide bonds reach the target cells or the target tissue and there, especially if they can not reach other cells in this region of the organism, completely before their transfection the target cell will be activated.

Due to the proposed highly selective effect of the molecules as a result of target cell-enzyme-specific inactivity / activation, the transfecting biologically inactive molecular constructs are administered systemically with appropriate peptide binding (with defined amino acid combinations).

The molecular constructs can also be bound to other substances (for example nanoparticles as a carrier system) for better transport into or onto the target cells and for their stabilization.

In addition, in order to improve the transfection rate, known mechanisms of action which increase the tissue or cell selectivity (for example ligand / antibody / antigen-labeled nanoparticles, TAT protein flanking) can be used. The combination with these known mechanisms of action supplement the described invention and lead to a reduction of the amount of active ingredient according to the invention to be used due to the preselection of the target cells. This results from the fact that in the introduction of active ingredient in vivo into the bloodstream, especially cells in well-perfused tissues are achieved. The proposed molecules with the biologically inactivating binding according to the invention to one or more peptides can be handled very well with transfection systems known per se, for example nanoparticles with or without ligand / antibody / antigen labeling, with enveloping nanoparticles or lipids and lipid-based transfection methods, be applied. Depending on their intended use, such transfection systems already reduce organ transplantation-related erroneous transfections, as a result of which, in relation to one another, cells are increasingly transfected in the target tissue and the quantities of active substance used can be kept to a minimum in each case. While this mis-transfection can not be prevented by use of the invention, the mis-transfected molecules in these other cells than the target cells, although still undesirable, are not biologically active. This status remains unchanged even in spite of inventive molecule activation in or on the target cells, so that the biological effect is selectively carried out exclusively in the target cells and in contrast to the known mechanisms a highly cell-selective modulation of gene expression is achieved.

The invention is not limited to applications for the treatment of tumors and, for example, virus (eg HIV) infected cells. The molecular constructs according to the invention can be used in all clinical pictures. which are based on increased or decreased gene expression, can be used to treat the disease itself or its symptoms. Furthermore, age or developmental changes in the gene expression pattern of cells can be treated. In addition, the invention may be used for any purpose whose purpose is to selectively alter the expression of genes in particular cells in vitro or in vivo.

Advantageous is an application kit with which the biologically active molecules to be used are provided with the bound peptides which, according to the proposal, have amino acid sequences selected with regard to the particular enzymes of the target cells, as well as a suitable transfection system and other additives. The application kit contains all necessary ingredients as well as an application manual for the intended use by a user.

The invention will be explained below with reference to exemplary embodiments illustrated in the drawing. Show it:

1 shows a general structure of the cell-specifically activatable molecular constructs according to the invention (inactivated state; in example a) the formation of the RISC is prevented by the altered spatial structure of the molecules and in example b) the binding to the niRNA is suppressed) a) Example of siRNA with two bound Peptides b) Example of a bound peptide PNA

2 shows an example of a siRNA molecule construct bound to a transfection system for the purpose of transfection into target cells,

Activation of this siRNA molecule construct a) Biologically inactive molecule construct for transfection into the target cell b) Binding disruption by the target cell specific enzyme c) Biologically activated molecule in or on the target cell with disrupted binding between the siRNA and the peptide

FIG. 3: siRNA molecule construct with two peptide bonds and a transfection system for transfection into target cells a) molecule construct with peptide bonds b) disruption of the peptide bonds by different enzymes of the target cell

Fig. 4: Representation of an exemplary compound between a peptide and a siRNA, a possible enzyme for cleavage of the peptide bond in this case caspase-4

FIG. 1 shows the general structure of the cell-specifically activatable molecular constructs according to the invention in the inactivated state by way of example for siRNA (FIG. 1 a) and for PNA (FIG. 1 b). In Fig. Ia, a siRNA 1 is bound as a biologically active molecule to two peptides 2, 3. With these bilateral peptide bonds, siRNA 1 is biologically inactive and is transfected into a target cell (not shown for reasons of clarity).

FIG. 1b illustrates how, instead of siRNA 1, a PNA 4 is bound to peptide 2 as a biologically active molecule and thus also biologically inactivated for transfection.

FIG. 2 shows a possible construct which could be used for transfecting the peptide binding (peptide 2) inactive biologically active molecule (siRNA 1) into a target cell. In this case (FIG. 2 a), antibody 6 for semi-selective binding to target cells, and also polyethylene glycol chains (PEG) 7 for anchoring peptide 2 and siRNA 1 could be bound to a nanoparticle 5.

Furthermore, the binding between the siRNA 1 and the inactivating peptide 2 is shown as an interface 8 for disruption by a selectively cutting and target cell specific enzyme 9 (Figure 2b). This enzyme 9, which only in or on the said (not If the target cell is present, the peptide bond of the siRNA 1 breaks down at the interface 8 (FIG. 2c) due to the specific amino acid sequence of the peptide 2. The biologically active molecule (siRNA 1), which is now again active due to the disrupted peptide bond, and the remaining construct consisting of the nanoparticle 5, the antibody 6, the polyethylene glycol chains (PEG) 7 and the peptide 2 dissolved by the siRNA 1, are thus separated. In or on other cells of the organism than the intended target cells into which the biologically inactive molecule construct (see FIGS. 2 a and 2 b) likewise transfects and in or on which the target cell-specific enzyme 9 is not present, the peptide bond of siRNA 1 remains obtained according to the invention at the interface 8. The siRNA 1 is still inactive as a biologically active molecule (see Fig. 2a). The intended in the target cells biological effect of siRNA 1 is not performed by the unbroken interface 8 in other cells.

In Fig. 3 a, an extension of the construct of Fig. 2 is shown. Again, in each case a peptide 2, 3 is bound to biological inactivation of the siRNA 1 at various points thereof (compare FIG. The various amino acid sequences in the bound peptides 2, 3 are selected so that two specific (or exclusively) specific enzymes 9, 10 present in or on the target cell can break up the peptide bonds of these cleavage sites 8, 8 ', respectively. If only one of the two peptides 2, 3 also does not exist in a cell other than the said target cell, into or onto the construct according to Fig. 3a (for example after miscoletion), at least one of the two remains Peptide bonds at the interfaces 8 and 8 'due to the lack of the equivalent target cell specific enzyme 9, 10 obtained. The siRNA 1 would continue to be inactive, even with a single remaining peptide bond. Exclusively at the target cell, in or at which the enzymes 9, 10 break through both interfaces 8, 8 'through the abovementioned defined amino acid sequences of the peptides 2, 3 (indicated in FIG. 3b), the siRNA 1 is separated from the remainder of the molecular construct (cf. also Fig. 2c). Thus, the siRNA 1 as a biologically active molecule only in this target cell unfold the intended purpose of the transfection effect.

Figure 4 shows a possible linkage between the siRNA 1 and a peptide having a possible specific amino acid sequence 11 (amino acid sequence is -L-E-V-D-) for a caspase-4 selectively cleaving enzyme present in a target cell. This would break the connection for the activation of siRNA 1 at the caspase-4 cleavage site symbolized by the arrow and designated.

In the example shown, after the cleavage by the enzyme caspase-4, a residue of the molecule would remain on siRNA 1, but this does not impair its biological activity.

The invention is not limited to the abovementioned amino acid sequence (-L-E-V-D-) shown in FIG. 4 for the disruption of the peptide bond on siRNA 1 by the target cell-specific enzyme caspase-4. In the following table, further amino acid sequences of the peptide for the proposed use for specific target cell enzymes are compiled by way of example:

Die vier in vorstehender Tabelle erstgenannten Zielzellenenzyme können auch auf der Oberfläche der Zielzelle bzw. in deren Umgebung vorkommen und wären in diesem Fall in der Lage, für eine solche Anwendung der biologisch wirksamen Moleküle die entsprechende Peptidbindung mit der erfindungsgemäß ausgewählten Aminosäuresequenz bereits unmittelbar vor der Transfektion in die Zielzelle aufzubrechen. Diese Verwendung wäre insbesondere denkbar, wenn die im inaktiven Zustand in den Bereich der Zielzelle gelangten biologisch wirksamen Moleküle nach ihrer gänzlich oder teilweise bereits außerhalb der Zielzelle erfolgten Aktivierung in diesem Teil des Organismus keine anderen (unerwünschten) Zellen mehr erreichen können, beispielsweise zur Transfektion in einen Zielzellenkomplex.

The four target cell enzymes mentioned in the above table may also be present on the surface of the target cell or in its environment and in this case would be able to use the corresponding peptide bond with the amino acid sequence selected according to the invention immediately prior to transfection for such application of the biologically active molecules to break into the target cell. This use would be conceivable in particular if the biologically active molecules which have reached the region of the target cell in the inactive state can no longer achieve any other (unwanted) cells after their activation in this part of the organism, either completely or partially already outside the target cell, for example for transfection into a target cell complex.

Advantageous is an application kit in which the biologically active molecules to be used are provided with the bound peptides which have amino acid sequences as proposed with regard to the particular enzymes of the target cells. The application kit should be in ampoule form containing all necessary ingredients, conveniently also a selection of suitable transfection systems (such as nanoparticles or lipids), other additives (such as antibodies, ligands and polyethylene glycol) and one or more probes or syringes with cannula for injection of the mixture of the Ampoule contents in containing the target cells containing medium. According to an enclosed application manual with a comparison of the selectable peptide-amino sequences to the respective enzymes of the target cells (see the above table), the user can assemble and apply appropriate application mixtures for the intended use.

It is advisable to provide such an application kit specifically for selected target cells and target genes as well as depending on the field of application (in vitro or in vivo). LIST OF REFERENCE NUMBERS

siRNA

2, 3 peptide

4 PNA

5 nanoparticles

6 antibodies, ligand

7 Polyethylene glycol (PEG)

8, 8 'interface (binding) between peptide and siRNA

9, 10 Enzyme of the target cell which cleaves the link between the peptide and siRJMA to activate it

11 amino acid sequence of the peptide (in the example: specific amino acid sequence for the enzyme

Caspase-4)

Claims

claims 1. Biologically active molecules, in particular based on PNA and siRNA, for biologically inactive transfection into a target cell, in order to the latter in this after biological activation by binding to mRNA and, for example, in the case of siRNA to form a "RISC complex to the expression of genes inhibit, characterized in that the biologically active molecules (1) for the purpose of their biological inactivation outside the target cell at least one or only by the cell-specific Enzymes (9, 10) of this target cell have breakable covalent bonds (8, 8 ') with at least one peptide (2, 3), each one or more enzymes selected for the target cell or enzymes and for the breakable covalent bond (8, 8 ') each having significant amino acid sequences (11). 2. Biologically active molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme caspase-4 has the amino acid sequence -L-E-V-D-. 3. Biologically active molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme matrix metalloproteinase-1, the amino acid sequence -Pro-Leu-AIa - Leu-Trp-Ala-Arg- contains. 4. Biologically active molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific , Enzyme Matrix metalloproteinases-2,7 has the amino acid sequence -Pro- Leu-Gly-Leu-Dpa- AIa-Arg-. 5. Biologically active molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of disruption of the binding (8, 8 ') by the cell-specific enzymes matrix metalloproteinases-2, 9 has the amino acid sequence -Pro-Leu-Gly-Met-Tφ-Ser-Arg-. 6. Biologically active molecules according to spoke 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme matrix metalloproteinases-3, 1,2,9 the amino acid sequence - Arg-Pro-Lys-Pro-Tyr-Ala-Nval-Trp-Met-Lys- has. 7. Biologically active molecules according to spoke 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme cathepsin S, the amino acid sequence -Phe-Arg-Phe ( p-nitro) - contains. 8. Biologically active molecules according to spoke 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme cathepsin-G, the amino acid sequence -Ala-Ala-Phe- contains. 9. Biologically active molecules according to spoke 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme cathepsin-D the amino acid sequence -Arg-Gly-Phe- Phe-Leu- includes. 10. Biologically active molecules according to spoke 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the line-specific enzyme angiotensin converting enzyme, the amino acid sequence -Gly-His- Leu. 11. Biologically active molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme Angiotensin converting enzyme having the amino acid sequence -Phe-Gly-GIy-. 12.Biologisch effective molecules according to claim 1, characterized in that the at least one peptide (2, 3) for the purpose of breaking the bond (8, 8 ') by the cell-specific enzyme angiotensin converting enzyme, the amino acid sequence -Gly-p- Nitro-Phe-Pro. 13. A method for cell-specific activation of biologically active molecules in or on a target cell, in particular based on PNA and siRNA, in which the biologically active molecules are preferably transfected biologically inactive in a target cell to there after their biological activation with binding to mRNA and, for example in the case of siRNA, with the formation of a "RISC complex to inhibit the expression of genes, characterized in that the biologically active molecule for Purpose whose biological inactivation outside the target cell or before transfection is covalently bound to one or more peptides each having at least one selected to the one or more target cell type enzymes and significant for the breakable covalent bond amino acid sequence and that the covalent binding of the peptide or to the biologically active molecule is broken for the purpose of its biological activation in each case by the target cell-typical enzyme. H. Process according to Claim 13, characterized in that the biologically active molecule is coupled to the covalently bonded peptide (s) for the purpose of improving transfection into target cells to a transfection system. 15. The method according to claim 14, characterized in that as a transfection system, a carrier system, for example nanoparticles with or without ligand / antibody / Antigenmarkierang, is used. 16. The method according to claim 14, characterized in that serve as a transfection system molecules, such as nanoparticles or lipids, with which the selectively activatable biologically active molecules are enveloped for their transfection. 17. Method according to claim 14, characterized in that lipid-based methods are used as the transfection system. 18. The method according to claim 14, characterized in that a TAT protein flanking is provided as the transfection system. 19. The method according to claim 13, characterized in that the biologically active molecule transfϊziert in the inactivated state in the target cell and is activated in this by the cell or the specific enzymes. 20. The method according to claim 13, characterized in that the biologically active, inactivated by peptide bond molecule immediately prior to transfection into the target cell by one or more on its surface or in their environment existing cell-specific enzymes (eg., Matrix Metalloproteinase- 1 , Matrix metalloproteinases-2, matrix metalloproteinases-3, matrix metalloproteinases-7, matrix metalloproteinases-9) partially or completely (based on the total number of covalent peptide bonds) is activated. 21. The method according to claim 13, characterized in that the biologically active molecule is coupled to the one or more covalently bound peptides to one or more marker substances. 22.Applikationskit for administering the biologically active molecules according to one or more of claims 1 to 21, consisting at least of at least one ampoule (ampoule A) containing one, for the purpose of inactivating a selected peptide with a specific one Amino acid sequence (for example, the amino acid sequence -Pro- Leu- Ala-Leu-Trp-Ala-Arg- for the target cell enzyme matrix metalloproteinase-1) bound siRNA contains and / or at least one vial (ampoule B), which one, for the purpose inactivation of a selected peptide with a specific one Contains amino acid sequence (for example the amino acid sequence -Pro-Leu-Ala-Leu-Trp-Ala-Arg- for the target cell enzyme matrix metalloproteinase-1) bound PNA, at least one further ampoule (ampoule C) with a transfection system, for example nanoparticles or lipids, Dilution and reaction buffer for the contents of ampoules A to C, - One or more probes or syringes with cannula and other materials required for injection of the mixture of the ampoule contents in the medium containing the target cells and instructions for administration with a comparison of the selectable peptide sequences for respective enzymes of the target cell. 23.Applikationskit according to claim 22, containing at least one substance, for example antibodies for the semi-selective binding of the biologically active molecules to target cells. 24.Applikationskit according to claim 22, containing at least one substance, for example polyethylene glycol chains, for anchoring the peptide and the biologically active molecules. 25. An application kit according to claim 22, containing at least one substance, for example lipid-based transfection reagent, for transfecting the biologically active molecules into the target cell. 26.Applikationskit according to claim 22, characterized in that it is provided for in vitro applications. 27.Applikationskit according to claim 22, characterized in that it is provided for in vivo applications.